Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (northwest Spain).

The purpose of this study was to establish the serogroups of Escherichia coli that cause avian colibacillosis in Spain. The serogroups of 625 avian E. coli isolated between 1992 and 1993 were determined. The 458 E. coli from chickens with septicaemia belonged to 62 different O serogroups; however, 59% were of one of 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116 and O132). These 18 serogroups were also determined as an important percentage (29%) of control isolates from faeces of healthy birds. Nevertheless, a significant difference (59% versus 29%; P < 0.001) was observed. Furthermore, the serogroups O12, O14, O18, O53, O78, O81, O102, O115, O116 and O132 were almost exclusively identified among septicaemic E. coli (31% versus 3%; P < 0.001). The high prevalence of O18, O81, O115, O116 and O132 isolates was not expected and may indicate the emergence of five new serogroups associated with avian colibacillosis not yet reported.

O serogroups, biotypes, and eae genes in Escherichia coli strains isolated from diarrheic and healthy rabbits.

A total of 305 Escherichia coli strains isolated from diarrheic and healthy rabbits in 10 industrial fattening farms from different areas of Spain were serotyped, biotyped, and tested for the presence of the eae gene and toxin production. The characteristics found in strains isolated from healthy rabbits were generally different from those observed in E. coli strains associated with disease. Thus, strains with the eae gene (74% versus 22%); strains belonging to serogroups O26, O49, O92, O103, and O128 (64% versus 12%); rhamnose-negative strains (51% versus 5%); and rhamnose-negative O103 strains with eae genes present (41% versus 1%) were significantly (P < 0.001 in all cases) more frequently detected in isolates from diarrheic animals than in those from healthy rabbits. Whereas a total of 35 serogroups and 17 biotypes were distinguished, the majority of the strains obtained from diarrheic rabbits belonged to only four serobiotypes, which in order of frequency were O103:B14 (72 strains), O103:B6 (16 strains), O26:B13 (12 strains), and O128:B30 (12 strains). These four serobiotypes accounted for 48% (112 of 231) and 5% (4 of 74) of the E. coli strains isolated from diarrheic and healthy rabbits, respectively. Only six strains were toxigenic (three CNF1+, two CNF2+, and one VT1+). We conclude that enteropathogenic E. coli strains that possess the eae gene are a common cause of diarrhea in Spanish rabbit farms and that the rhamnose-negative highly pathogenic strains of serotype O103:K-:H2 and biotype B14 are especially predominant. Detection of the eae gene is a useful method for the identification of enteropathogenic E. coli strains from rabbits. However, a combination of serogrouping and biotyping may be sufficient to accurately identify the highly pathogenic strains for rabbits.

Prevalence and characteristics of human and bovine verotoxigenic Escherichia coli strains isolated in Galicia (north-western Spain).

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H-, 091:H21, O103:H2, 0105:H18, O111:H-O113:H21, O126:H-, O128:H- and O157:H7 or H-) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (O26:H11 and O86:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes. 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1+ strains of serotype O26:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (O26:H11, O111:H-, O157:H-and O157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.

Penner serotyping of Campylobacter isolates from poultry, with absorbed pooled antisera.

The Penner serotyping system, based on detection of heat-stable antigens with a passive haemagglutination technique, was used in studies on Campylobacter epidemiology in poultry. Preparation of specific antisera by absorption allowed the use of pooled antisera. Over 80% of the Campylobacter isolates were typable with this modified Penner serotyping system. Typability of strains was clearly affected by storage of the strains before actual typing. Extracted antigens appeared to be stable for at least 6 months at 4 degrees C. Therefore, it is advisable to store extracted antigens from freshly isolated Campylobacter strains instead of reculturing frozen-stored strains, when actual typing cannot be performed directly after primary isolation. Untypability of isolates may partly be explained by the detection of Campylobacter serovars not yet represented in the serotyping system. Experiments on repeated serotyping of several Campylobacter strains did not suggest any serovar instability within the strains.

Serotypes of CNF1-producing Escherichia coli strains that cause extraintestinal infections in humans.

The O:K:H serotypes of 137 necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.

Serotypes of bovine Escherichia coli producing cytotoxic necrotizing factor type 2 (CNF2).

The serotypes of 101 faecal bovine necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factor type 2 (CNF2) were determined. Although, NTEC producing CNF2 belonged to 48 different O:K:H serotypes, only eleven of them accounted for 54% of strains. The most common serotypes in order of frequency were: O123:K-:H16, O3:K-:H21, O88:K-:H8, O15:K14:H21, O1:K-:H12, O1:K1:H46, O2:K1:H5, O55:H21, O88:K?:H25, O117:K?:H21 and O123:K-:H-. The serotypes of CNF2 NTEC were different from those found in NTEC producing CNF1 and in enterotoxigenic, verotoxigenic, enteropathogenic, enteroinvasive and enteroadherent E. coli strains that cause infections in humans and animals.

PCR-mediated DNA typing of Campylobacter jejuni isolated from patients with recurrent infections.

The polymerase chain reaction (PCR) and two primers aiming at the enterobacterial repetitive intergenic consensus (ERIC) and arbitrary DNA sequences, respectively, were used to fingerprint the genomic DNA of 24 Campylobacter jejuni strains isolated from five patients with recurrent C. jejuni infections. Results were compared with biotyping and serotyping. The latter two methods, when combined, distinguished 9 different types, whereas PCR-mediated DNA analysis discriminated 14 different patterns. For six strains, the results of PCR-mediated typing led to different interpretations. This method is proposed as an additional tool to further discriminate between C. jejuni strains that appear related by conventional typing methods. In view of its rapidity and simplicity, this method is a potential candidate to replace the relatively slow and laborious conventional methods. However, further study is needed to assess the sensitivity and specificity of PCR-mediated DNA analysis and to investigate the usefulness of this method as an epidemiological tool in outbreaks of Campylobacter infections.

Serotypes and colonization factors of enterotoxigenic Escherichia coli isolated in various countries.

One hundred and six enterotoxigenic E. coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity. CFA/I was found in 6 (17%) of 36 LT+STa+ strains and in 15 (54%) of 28 STa+ strains; CFA/II was found in 16 (44%) of 36 LT+STa+ strains. None of 42 LT+ strains showed CFA/I or CFA/II. CFA/I was found in ETEC of serotypes O63:K-:H-, O78:K80, O128:K67 and O153:K:H45, whereas CFA/II was found in serotypes O6:H-, O6:K15:H16 and O6:K?:H40. Of the 69 CFA/I- CFA/II- ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic. Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT+ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT+ and 1 LT+ STa+) also negative for MRHA, and (iii) 3 STa+ strains of serotype O27:K-:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is. The 106 ETEC strains belonged to 20 different O serogroups. However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167). E. coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples.

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv-locus in all Yersinia spp. and the presence of the ail-gene in pathogenic Y. enterocolitica only. Hybridization results with ail-probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI+ strains and only two TCI- strains were positive by hybridization with ail. Hybridization results with inv- or ail-probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv- or ail-probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv- and/or ail-probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv- and/or ail-probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.

Frequency of enterovirulent Escherichia coli in diarrhoeal disease in The Netherlands.

To assess the role of enterovirulent Escherichia coli in The Netherlands, faecal samples of 279 patients (108 children, 171 adults) with diarrhoea and 100 healthy controls were investigated in a prospective study. Enterovirulent Escherichia coli were identified by hybridization with five different non-radioactively labelled DNA probes specific for enteropathogenic Escherichia coli (EPEC), verocytotoxin producing Escherichia coli (VTEC) and enterotoxigenic Escherichia coli (ETEC). The rate of isolation of EPEC was 6.5% in patients with diarrhoea and 2.0% in asymptomatic persons. During the study period, no VTEC were isolated from patients with diarrhoea. ETEC were isolated from two persons, both of whom had experienced diarrhoea and had returned from travel in (sub)tropical areas. Our results suggest that diarrhoea is sporadically caused by ETEC among the indigenous population of The Netherlands, and is mainly associated with travel in endemic areas. Furthermore, the presence of EPEC probe-positive strains in the stool need not always be accompanied by symptoms of diarrhoea.

Bovine Escherichia coli of serotypes O55:H4 and O55:H21 which produce CNF2 express P fimbriae and Vir surface antigen, respectively.

We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55. Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans. In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen. One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination.

Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model.

Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.

Typing of Aeromonas strains from patients with diarrhoea and from drinking water.

Aeromonas strains (187) from human diarrhoeal stools and from drinking water (263) in The Netherlands were typed by three different methods. Biotyping alone was found to be of little value for epidemiological studies because 84% of all strains belonged to only 10 biotypes. Common biotypes could be further differentiated by serotyping. Gas-liquid chromatography of cell wall fatty acid methyl esters (FAME) was useful for species identification as well as for typing: 86% of all strains could be identified to the species level, and within this group 92% of all identifications corresponded with the biotype. Cluster analysis and principal component analysis of FAME profiles could be used for comparison of strains from different sources and gave the same general conclusions as bio- and serotyping. There was little overall similarity between Aeromonas strains from human (diarrhoeal) faeces and from drinking water, differences being most pronounced for Aeromonas caviae and least for A. sobria.

Construction and analysis of Bordetella pertussis mutants defective in the production of fimbriae.

Although the role of fimbriae in bacterial disease has been well established, little is known about the function of Bordetella pertussis fimbriae. To study this function, well-defined fimbrial mutants were constructed. B. pertussis harbours three fimbrial genes, fim2, fim3 and fimX, and strains were constructed in which one or more fimbrial genes were inactivated by means of gene replacement. Analysis of these strains by means of immunoblotting suggested the presence of a fourth fimbrial gene, tentatively designated fimY. A fimbrial mutant was analysed in a mouse respiratory infection model, together with a strain harbouring a deletion in the gene for the filamentous haemagglutinin. Both mutants were affected in their ability to persist in the trachea. Persistence in the nasopharynx was only affected by the mutation in the filamentous haemagglutinin gene. Neither the filamentous haemagglutinin nor the fimbrial mutants were affected in their ability to persist in the lung. Our results suggest that the filamentous haemagglutinin plays a more crucial role than fimbriae in the colonization of the upper respiratory tract of the mouse.

Analysis of Escherichia coli isolates from subjects with travellers' diarrhoea using DNA probes and serotyping.

Escherichia coli isolated from faeces of 54 healthy volunteers who visited Tunisia for eight days were examined. These volunteers participated in a randomized double-blind placebo-controlled study to establish whether ciprofloxacin could prevent travellers' diarrhoea. Escherichia coli strains isolated before travel, during episodes of travellers' diarrhoea, immediately after return and five weeks after return were serotyped and tested for the presence of virulence genes indicating diarrheogenic properties by hybridization with a set of four non-radioactively labelled DNA probes. Subjects receiving ciprofloxacin prophylactically to prevent travellers' diarrhoea were asymptomatic and no Escherichia coli could be cultured shortly after return home. Sixty-four percent of subjects (18/28) who did not receive antibiotic prophylaxis suffered from travellers' diarrhoea. Hybridization tests detected 8 enterotoxigenic Escherichia coli strains producing heat stable toxin, 13 enterotoxigenic strains producing heat labile toxin and 10 strains which produced both heat labile and stable toxin. Of the 31 probe positive strains, 29 (94%) were cultured from 11 volunteers with travellers' diarrhoea. A bacterial cause was thus determined in 61% of the volunteers who experienced travellers' diarrhoea.

Quinolone resistance in campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine.

Eight hundred and eighty-three strains of Campylobacter spp. isolated between 1982 and 1989 from human stools and poultry products were screened for quinolone resistance. In this period the prevalence of resistant strains isolated from poultry products increased from 0% to 14%. During the same period the prevalence in man increased from 0% to 11%. The emergence of quinolone resistance has implications for the identification of campylobacter up to species level: the susceptibility for nalidixic acid can no longer be used as a criterion for identification in the laboratory. The rapid emergence of resistant campylobacter may also have important implications for the treatment and prophylaxis of diarrhoeal disease. The increase of quinolone resistance coincides with the increasing use of fluoroquinolones in human and veterinary medicine. Extensive use of enrofloxacin in poultry and the almost exclusive transmission route of campylobacter from chicken to man, in The Netherlands, suggests that the resistance observed is mainly due to the use of enrofloxacin in the poultry industry.

Salmonella contamination of poultry flocks in The Netherlands.

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.

Prevalence of enterotoxigenic Escherichia coli strains in outbreaks and sporadic cases of diarrhoea in Spain.

Escherichia coli strains isolated 1985-1988 in Spain from patients with diarrhoea were examined; 1170 strains were isolated from 582 sporadic cases of diarrhoea in children, and seven strains were associated with seven outbreaks of diarrhoea. Strains positive for STa enterotoxin production in the infant mouse test were also assayed for production of LT enterotoxin on Vero cells and by a coagglutination test. Thirty-one strains were STa positive: 28 were isolated from 16 (2.7%) sporadic cases of diarrhoea and three were responsible for outbreaks. The majority of STa+LT- strains from both outbreaks and sporadic cases were serotype O153:H45 and expressed the CFA/I colonization factor antigen. Enterotoxigenic STa+LT- strains of serotype O27:H7 and STa+LT+ CFA/II+ strains of serotype O6:K15:H16 were also isolated frequently from sporadic cases.

Infection and immunity to Vibrio cholerae, Salmonella typhimurium and Escherichia coli in a rabbit model.

Cholera disease can be induced in the rabbit by duodenal inoculation (DI) of Vibrio cholerae organisms after ligation of the cecum (C) (DIC model). When ligation of the cecum is omitted, no disease symptoms develop. In contrast, the animals are primed which becomes apparent as vibriocidal protection upon challenge with V. cholerae in the DIC model. This protection coincides with high anti-O antigen IgA levels in the bile. The O antigen was shown to be the protective antigen and it must be presented by live organisms. A non-enterotoxigenic mutant of V. cholerae induced protective immunity in the rabbit but was reported to cause mild diarrhea in human volunteers. Looking for alternatives, we applied cholera toxin, known as a mucosal adjuvant, together with killed V. cholerae cells to rabbits. Unfortunately, the minimum adjuvant dose was equal to the minimum toxic dose. A Salmonella typhimurium strain expressing also the V. cholerae O antigen induced systemic rather than local immunity which was not protective. Several Escherichia coli strains were able to elicit a local immune response, but the animal to animal differences were considerable. Therefore, V. cholerae itself was thought to be the most appropriate carrier organism. Some non-enterotoxigenic and auxotrophic mutants of V. cholerae were able to prime and did not show any undesired side-effects in the DIC model. Therefore, further attenuation of non-toxigenic V. cholerae strains by means of stable deletions in nutritional genes seems to be the most promising way to obtain acceptable vaccine candidates.

Value of the Widal test in the diagnosis of typhoid fever in an endemic area and suggestions for a modification. A preliminary study.

The Widal test for detection of typhoid fever was introduced in two rural hospitals in Nigeria. Because of undetectably low O agglutination titers, 74 sera were sent overseas for further determination by means of passive haemagglutination with O 9, 12-antigen, Vi-antigen and slide agglutination with H-d antigen suspensions. This full test showed discrepancies with the normal Widal test i.e. a false negative Widal H agglutination in 6 out of 17 patients in West Nigeria and a false positive Widal H test in 28 out of 49 patients in Central Nigeria. The full test should be applicable in a small laboratory, but its value has yet to be established.